ABSTRACT Over the past four decades, endothelial cells lining the vessel wall have become an intense subject of clinical interest since ease of accessibility allows for effective drug targeting. Dysregulation of vessel growth is associated with wide range of disease states such as diabetic retinopathy, wet form of age related macular degeneration, and tumor growth. Tumor growth is angiogenesis dependent. The first step in tumor angiogenesis is endothelial cell activation and directional migration of endothelial cells towards the tumor. Therefore, understanding the mechanisms of directional migration is critical for development of therapeutics that can selectively target tumor endothelial cells from taking this first step. The goal of this laboratory is to identify and study candidate genes that are selectively upregulated in tumor vasculature. Robo4 is an excellent target since it is expressed selectively in tumor vessels and is critical for directional migration of endothelial cells. Further, the cell surface expression of Robo4 allows easy access to potential drugs. Importantly, specific signaling complex assembly on Robo4, if identified to be responsible for directional migration can also be targeted. Previously, we have shown that robo4 is essential for angiogenesis in vivo in zebrafish and activates Rho GTPases in endothelial cells in vitro. Preliminary results suggest that: (A) Robo1 and Robo4 interact and share common signaling molecules at both the cell surface and intracellular level; (B) A complex of proteins including IRSp53, Mena, Cdc42-GTP, and Vilse together assemble in endothelial cells in response to Robo4; (C) Members of the Sry-related high-mobility-group (Sox) family interact with Robo4's cytoplasmic tail. We hypothesize that: Endothelial cell surface receptors Robo1 and Robo4 co-operatively interact with Slit2 to direct cell migration via a specific complex assembly of intracellular signaling proteins (Cdc42-GTP, Vilse, IRSp53, Mena, Sox). To test this hypothesis in aim 1, we will investigate whether Robo1 and Robo4 co- operatively interact with Slit2 and direct signaling complex assembly of Vilse and Mena in endothelial cells using a combination of molecular, biochemical and imaging methods. In aim 2, we will identify the mechanism of assembly of IRSp53, Mena, Cdc42-GTP, Vilse and Sox with Robo1 and Robo4's cytoplasmic tail in a signaling complex responsible for directing endothelial cell migration using a combination of in vitro biochemical and in vivo functional assays in zebrafish. We anticipate that by accomplishing aims 1 and 2, we will understand how Robo1 and Robo4 dictate the endothelial tip cell to navigate through complex milieu by unraveling the similarities between the two molecules in triggering signaling in endothelial cells, the order in which the different signaling molecules assemble on Robo1 and Robo4's cytoplasmic tail, the cues they respond to, and the integration of different signaling components to form filopodia. Each mechanistic step is a putative target and drugs targeting these steps will benefit diseases characterized by excessive angiogenesis and vascular leakage.